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Medical Microbiology. Disinfection and Sterilization. —— For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU September 18, 2007 Mengfeng Li ( 黎孟枫) , M.D. Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

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Disinfection and Sterilization

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Medical Microbiology

Disinfection and Sterilization

——For the course of Medical Microbiology for MBBS foreign students, Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li (黎孟枫), M.D.

Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

[email protected]

Early needs and success for disinfection (Early 1800’s) —— a historic story

  • By mid-1800’s, almost half of post-operative patients died of sepsis (then called “hospital disease”). A common report by surgeons was: operation successful but patient died.

  • A hypothesis: ① exposing moist body tissue to oxygen  sepsis;② best prevention: keeping air away from wounds by means of plasters, collodion or resins.

  • Having tried methods to encourage clean healing with no success, surgeon Joseph Lister discarded the concept of direct infection by bad air but postulated that sepsis might be caused by a 'pollen-like dust‘, although he did not know yet the “dust” was living microbes.

  • When Louis Pasteur suggested the presence of living organisms in the air, Lister made the connection with wound sepsis: the microbes in the air were likely causing the sepsis and should be destroyed before they entered the wound.

  • Lister had previously heard that 'carbolic acid' was used to treat sewage, and that fields treated with the affluent were freed of a parasite causing disease in cattle. He then began to clean wounds and dress them with carbolic acid..

The milestone:

case of James Greenlees

-Aug. 12, 1865, James Greenlees, 11-year-old

-Struck by a horse-drown cart

-Compound fractures

-Taken to Royal Infirmary

-Under the care of Joseph Lister

Joseph Lister operated with carbolic acid (石炭酸)spray

Why disinfection and sterilization?

  • Contagious diseases

  • Hospital infection (e.g., OR, ID ward) or other opportunistic infection

  • Lab contamination

  • Etc.

  • Microbes:

  • usually easy to grow in environment;

  • but also can be inhibited or killed by certain environmental (physical or chemical) factors/conditions.


  • Disinfection消毒

  • Sterilization灭菌

  • Bacteriostasis抑菌

  • Antisepsis防腐

  • Asepsis 无菌


  • Process of reducing or eliminating living pathogenic microorganisms in or on materials, so they are no longer a health hazard.

    For example: use of alcohol before drug injection.


  • Process of destroying all microbial forms. A sterile object is one free of all microbial forms, including bacterial spores.

  • More thorough than disinfection


  • Process of inhibiting the growth of microorganisms, in vivo (mostly) or in vitro.

    For example: bacteriostatic antibiotics

Antisepsis (防腐)

  • Process of inhibiting or preventing growth of microbes, mostly in vitro and not bactericidal or sporicidal (杀芽胞的 )

    For example: use of chemical agents on skin, other living tissues or food/beverage.


  • A state where no living microorganism exists.

    For example: OR (Operating Room)

Controlling Microorganisms with Physical Conditions

  • High Temperature (heat)

  • Radiation

  • Ultrasound

  • Filtration

  • Low Temperature

  • Desiccation(干燥)

static action

cidal action

High Temperature ——Dry heat and Moist heat  protein denaturation and clotting; DNA strand breakdown


Dry heat:protein oxidation

  • Incineration(焚烧)

    • most thorough (>500℃)

    • disposals and corpes

  • Flaming (burner)(烧灼)

    • test tube opening, transferring loop

  • Hot air sterilization/Baking(干烤)

    • 160-170℃, 2h

    • Glassware, china, syringes, needles, etc

  • Infrared heat: similar to baking

Moist heat:denaturing proteins and melt lipids

  • Autoclaving(高压蒸汽灭菌)

    • Most commonly used and effective

    • 121℃ (103.4kPa), 15-20min

    • killing both vegetative organisms and


  • Boiling

    • 100 ℃ (105℃ with 2% Na2CO2) , 15-20min

    • cidal for vegetative cells but not necessarily spores

  • Regular Steam (Arnold Sterilizer)

    • 100 ℃, 15-20min

    • cidal for vegetative cells but not necessarily spores

  • Pasteurization(巴氏消毒法)

    • to kill pathogens in readily perishable objects (milk, wine)

    • flash method (瞬间法): 71.6℃, 15s

    • holding method (持续法): 62.9℃, 30 min

  • Fractional sterilization(间歇蒸气灭菌法)

    • alternating exposure and cooling time for a consecutive period:

      Steam heating (100℃, 30 min)30℃ for endospores to germinate 100℃, 30 min to kill germinated endospores 30-37℃ overnight for remaining endospores to germinate 100℃, 60 min to kill last remaining germinated endospores

    • for sugar- or milk-containing culture media

Moist Heat vs Dry Heat

Moist heat Dry heat

Penetrating potency higherlower

Temp for protein clotting lowerhigher

Extra heat released yesno

from condensation

Sterilizing potency: Moist heat >> Dry heat


  • Ultraviolet (UV) radiation

    • mechanism: blockage of DNA replication by forming thymidine dimmers

    • microbicidal activity of UV depends on:

      • length of exposure

      • wavelength: 200-300 nm, with the best effect of 265-266nm

      • bulb life (4000hr)

    • very poor penetrating power

    • for air or surface disinfection

      (OR, ID ward, labs)

    • causing eye damage, burns

      and mutation in skin cells

  • Ionizing Radiation(电离辐射)

    • X-rays, gamma rays and high-speed electrons

    • generating more energy and penetrating power than UV

    • to sterilize pharmaceuticals, disposable medical supplies (e.g., syringes, gloves, catheters, sutures) and foods

  • Microwave

    • penetrating non-metal materials (glass, plastics, china)

  • Ultrasound

    • more effective for gram-negative bacteria

    • Lack of thoroughness —— survivors remain

  • Filtration

    • sterilize heat- or chemical-sensitive solutions

    • not effective for virus, ricketia, mycoplasma

Seitz filter

  • Desiccation

    • static effect byinhibiting microbial enzymes

    • not effective against endospores

    • mainly for food reservation

  • Low Temperature (-20℃ ~ -70℃)

    • inhibits microbial growth by slowing down microbial metabolism

    • a special form: lyophalization (freeze-drying), used for long-term (years) reservation of bacteria stocks

      • fast freezing + drying

      • protecting agents (glycerol, serum)

Control Microorganisms with Chemical Agents (Disinfectants and Antiseptics)

Antimicrobial modes of action of disinfectants and antiseptics

  • Denaturation of bacterial proteins by disrupting hydrogen and disulfide bonds

    —— phenol (high conc.), alcohol, heavy-metal (high conc.), acids, alkalies, aldehydes)

  • Damage to bacterial membrane (lipids and/or proteins), causing leakage of intracellular molecules

    —— phenol (low conc.), surfactants, dyes

  • Interference of bacterial enzyme and metabolism

    —— oxidants, heavy-metals (low conc.), alkylating agents

  • Phenol and phenol derivatives

    ——altering membrane permeability and denaturing proteins

    • 0.01% - 0.05% Chlorhexidine(洗必泰)—— vaginal wash, OR hand-wash

    • 3% - 5% carbonic acid or 2% Lysol ——floor or surface disinfection

  • Alcohols

    —— denaturing bacterial proteins and membranes

    • 70% - 75% ethyl or isopropyl alcohol(乙醇或异丙醇)—— skin and thermometer disinfection

      • ineffective against endospores and non-enveloped viruses

  • Heavy metals (Hg2+、Ag+)

    —— denaturing proteins and inactivating enzymes

    • 2% mercurochrome(红汞)or 0.1% merthiolate(硫柳汞)—— skin, mucosa and wound

      • bacteriostatic, ineffective against endospores

    • 1% silver nitrate(硝酸银)—— eye drops for newborns to prevent gonococcal ophthalmia(淋菌性眼炎)

  • Oxidants

    —— oxidation, protein precipitation

    • 0.1% potassium permanganate(高锰酸钾)—— skin, fruits/vegetables

    • 3% peroxide(过氧化氢)—— small trauma wound, skin, mucosa

    • 0.2% - 1% peroxyacetic acid(过氧乙酸)—— plastics, glassware

    • 0.2 – 0.5 ppm cholorines —— water and swimming pool

  • Surfactants

    —— damaging bacterial membranes, inactivating enzymes, protein precipitation

    • 0.05-0.1% bromogeramine (新洁尔灭)—— OR hand-wash, skin, surgical instruments

  • Alkylating agent(烷化剂)

    —— alkylating proteins and nucleic acids

    • formalin (formaldehyde) ——surface disinfection, air, surgical instruments

    • glutaric dialdehyde(戊二醛)—— high-precision instruments, endoscopes

    • 50mg/L epoxy ethane(环氧乙烷)——surgical instruments and dressing

  • Dyes

    —— inhibiting bacterial growth by interfering with oxidation

    • 2% - 4% methyl violet(龙胆紫)—— wound disinfection

  • Acids and alkalies

    —— destroying cell membrane and cell wall, denaturing proteins

    • 5-10ml/m3 acetic acid evaporation ——air disinfection

    • quicklime [Ca(OH)2] ——floor and excretion (feces, urine, sputum, pus) disinfection

Effectiveness of antimicrobial agents are affected by ——

  • The concentration/intensity and nature of the disinfectant;

  • Length of exposure;

  • Species and number of the microbe(s);

  • Temperature and humidity;

  • Acidity (pH);

  • Presence of organic substances;

  • Presence of chemical antagonists

  • The nature of the material bearing the microbes

Summary 1. Application of chemical disinfectants

Patient excretionChlorines, 5% carbonic acid, 2% Lysol

Skin (hands)2% Lysol, 0.2-0.4% peroxyacetic acid(过氧乙酸)for HBV, 70% ethyl alcohol, 2% mercurochrome(红汞)

Mucosaoral- 3% peroxide; uri-reproductive- 0.01-0.05% Chlorhexidine(洗必泰), 0.1% potassium permanganate(高锰酸钾); newborn eyes- 1% silver nitrate

Drinking waterChlorines

Toilets, sewagequicklime [Ca(OH)2]

Air (OR, ID ward)formalin steam (12.5-25ml/m3,12-24h),formalin 40ml + potassium permanganate 30g/m3;

HBV ward- peroxyacetic acid 3g/m3 90min

Glassware, china,0.5% iodophores, 0.2-0.4% peroxyacetic

Rubber, metal acid


Summary 2. Potency levels of chemical disinfectants

Potency DefinitionExamples

High Killing all microbes including glutaric dialdehyde(戊二醛),

endospores and TBformaldehyde peroxyacetic acid(过氧乙酸),epoxy ethane(环氧乙烷)

Medium Killing all non-spore microbesalcohol, chlorines, iodophores

including TB

Low Killing vegetative bacteria chlorhexidine(洗必泰),

and lipophilic (enveloped) bromogeramine(新洁尔灭)

viruses, but resisted by

endospores, TB and hydrophilic

(non-enveloped) viruses

Summary 3. Spore-killing effects of chemical disinfectants

Spore-killing disinfectins

  • glutaric dialdehyde(戊二醛), formaldehyde(甲醛), Iodines, H2O2, epoxy ethane(环氧乙烷)

    Non spore-killing disinfectins

  • alcohols, phenols, chlorhexidine(洗必泰),


Medical Microbiology

Disinfection and Sterilization

——For the course of Medical Microbiology for MBBS foreign students,Class 2006/2011, SYSU

September 18, 2007

Mengfeng Li (黎孟枫), M.D.

Department of Microbiology, Zhongshan School of Medicine, SYSU, Guangzhou, China

[email protected]

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